Our previously-published work has established that nitisinone (NTBC) can increase melanin pigmentation in a mouse model of OCA1B, but not OCA1A. Since the time of the last report, we have focused on the following sub-projects: 1. Visual function in NTBC-treated OCA1B mice vs. controls Based on the success with increasing pigmentation in OCA1B adult mice and in pups treated in utero, we asked whether we could detect eye morphological, cellular and functional changes in the latter after weaning. Through collaborations with the NEI Visual Function Core and the Neurobiology, Neurodegeneration & Repair Laboratory, we have studied the effect of prenatal NTBC treatment on neural retina development, neural circuit development and spatial visual acuity. Preliminary data on a small cohort of mice indicated that prenatal treatment of OCA1B mice with NTBC results in measurable changes in electroretinogram (ERG) and direct coupled (dc)-ERG responses that returned within normal values in the treated mice. However, no change in photoreceptor cell number, accessory optic tract routing, nor visual behavior measured by optokinetic and optomotor response was observed. While photoreceptor function measured by ERG seems to be preserved in OCA1B mice, behavioral measures such as optomotor response seem to indicate that the mice are blind. We are pursuing further studies in the OCA1B mice as well as in mouse strains in which pigmentation of the iris is preserved but not pigmentation of the RPE to test the hypothesis that iris pigmentation is a confounding factor in the interpretation of optomotor responses in OCA1B mice. 2. Effect of NTBC on a mouse model of OCA3 We hypothesized that NTBC might improve melanization in a mouse model of OCA3 (the Tyrp1-brown mouse), as tyrosinase (Tyr) and Tyrp1 are known to interact and stabilize one another in the melanosome membrane. The mice received from Jackson Labs (C57BL/6J-Tyrp1b-J/J) are homozygous for a novel mutation in Tyrp1 that we have characterized. They respond to NTBC treatment with increased plasma tyrosine concentrations and no overt toxicity. However, no obvious change in the color of fur or pigmented ocular structures could be discerned with the exception of the iris, suggesting that treatment of OCA3 patients with NTBC is unlikely to be therapeutic. The manuscript is in publication. 3. Effect of NTBC in mouse models of OCA 2 and OCA4 By analogy, we hypothesized that NTBC might improve melanization in mouse models of OCA2 (melanocyte-specific transporter protein) and of OCA4 (the so-called underwhite allele of SLC45A2). Similar to the OCA3 mice, plasma tyrosine concentration was increased in NTBC-treated OCA2 and OCA4 mice with no overt toxic side-effects. Unlike the OCA3 mice, fur pigmentation was augmented in both mouse lines. Iris pigmentation was augmented in both OCA2 and OCA4 mouse lines, with a more predominant effect in OCA4 mice. Electron microscopy also confirmed a small increase in pigmentation of the choroid in treated OCA2 and OCA4 mice whereas, no effect was observed in the RPE in both lines. A manuscript describing these data is currently being prepared for submission and publication. 4. High-throughput drug screening to find compounds that regulate Tyr activity In previous published work, we had purified recombinant wild-type and mutant human Tyr and established a fluorometric assay for measuring Tyr activity as a read-out for screening inhibitors and enhancers. In addition, we have recently demonstrated that full-length and truncated Tyr have similar enzymatic activities (Dolinska et al., PCMR, 2017), thus validating the use of the truncated protein in our high-throughput drug screening. In collaboration with NCATS, we successfully screened 34,000 compounds from the Genesis Drug Collection, the Natural Products Library, and the NCATS Pharmaceutical Collection. We identified new inhibitors (>100) and several activators of tyrosinase. After validation in a more detailed, secondary enzymatic screen in vitro, and in vivo in zebrafish, we have recently initiated the treatment of a mouse model of OCA1B with the top candidate compound. Safety as well as efficacy in increasing pigmentation in the fur and ocular structures are being assessed. 5. Clinical Protocol for Studying the Effect of Nitisinone Treatment in Human Subjects with OCA1B We have established an IRB-approved protocol for the testing of a standard oral dose of nitisinone on ocular and systemic melanin pigmentation in patients with OCA1B. Five patients were treated with nitisinone. Similar to the mouse, administration of nitisinone increased tyrosine plasma concentration in all subjects. Visual acuity was not altered by nitisinone treatment in adults, as expected in a mature visual system. Changes in iris transillumination, hair and skin pigmentation are now being analyzed.